Quorum sensing describes the cell-signaling systems that facilitate intercellular interactions among bacteria and coordinate population-level behaviors. Quorum sensing typically alters cellular behavior by regulating gene expression. Bacteria also use quorum sensing to regulate factors important for infecting humans and other animals. There is a gap in understanding how quorum-sensing systems are integrated into the infection process. Addressing this knowledge gap is important to gain basic insight into how pathogens and beneficial microbes form stable host-microbe associations, which will inform strategies designed to control these associations through modulating quorum sensing. The overall objective of this proposal is to determine how the bacterium Vibrio fischeri uses gene-regulatory networks associated with quorum sensing during infection of its natural host, the Hawaiian squid. V. fischeri infections initially assemble within the light organ of juvenile animals, which are un-colonized at the time of hatching. V. fischeri uses quorum sensing to regulate bioluminescence, which is its symbiotic function within the squid host. The genes necessary for bioluminescence are regulated by quorum sensing. The small RNA Qrr1 is a component of this regulatory network and negatively controls bioluminescence. Guided by preliminary data, this proposal investigates how V. fischeri uses this network during host colonization by addressing three aims: 1) Understand regulation of qrr1 transcription in V. fischeri, 2) Examine V. fischeri aggregates for phenotypic heterogeneity, and 3) Evaluate how aggregation during symbiosis establishment impacts the quorum-sensing pathway. All three aims will take advantage of the innovative platform afforded by the symbiosis: fluorescence microscopy will be used to directly examine the infection process and molecular biology tools to dissect the associated quorum- sensing systems. The proposed research is significant because it will reveal important basic principles of how quorum-sensing networks function at different infection stages, which is knowledge that has broad applicability to other host-microbe interactions and provides critical insight into strategies designed to control these associations through modulating quorum sensing.